When the supply of oxygen to the heart is interrupted (anoxia) or heart muscle is deprived of circulation (ischemia), re-establishment of the supply of oxygen (reperfusion) induces massive damage to the tissue. Ischemic or anoxic episodes are associated with diminution of ionic gradients across cell membranes and the generation of toxic metabolic byproducts. Heart cells, under physiological conditions, are extensively coupled by gap junctions permeable to substances up to I kD in molecular weight. Without compensatory mechanisms to limit the intercellar spread of ischemic- and reperfusion-induced damage, cell death might propagate throughout the entire heart. Animal experiments and clinical experience show that when localized regions of the heart become ischemic or anoxic, reperfusion damage is primarily limited to that tissue where circulation failed and was re-established. What specific cellular mechanisms localize ischemic or anoxic damage in heart? The goal of this application is to determine the role that gap junctions might play in limiting the spread of ionic concentration changes, depolarizations, and cell damage during ischemia and reperfusion. We will first assess gap junctional patency during deprivation by measuring the intercellular spread of fluorescent dyes in papillary muscle fiber preparations placed in a two compartment chamber, one side of which can be deprived of oxygen and nutrients. Intracellular calcium (Cai), potassium (Ki), sodium (Nai), and H+ (pHi) ion concentrations will be measured photometrically on the deprived and non-deprived sides of the preparation and related to reductions in intercellular dye coupling. The extent of deprivation will be assessed by measuring myoglobin absorbance or intrinsic NAD(P)H fluorescence. Measurement of dye uptake from the extracellular medium of the non-ischemic side will assess the spread of cellular damage to uninjured cells. The effects of changes in Cai, pHi, Ki, and Nai on junctional conductance (gj) will be quantitatively measured in voltage clamped pairs of isolated myocytes and compared with ionic changes and the reduction in dye coupling seen in the deprived tissue preparation. The role of second messengers such as CAMP, cGMP, and IP3 in mediating a gap junctional response to ischemia will be explored. The possible relationship between intracellular high energy phosphates (HEP) and gj will be tested by altering the intracellular concentrations of HEP, while controlling intracellular ion concentration changes. Partial intracellular dialysis of one cell of a myocyte pair will allow comparison of the effects of ionic or HEP concentration changes imposed on one side of the junction with those imposed bilaterally. This will be facilitated by extracellular interventions, dialysis with patch electrodes, and the measurement of ions and NAD(P)H/NAD(P) in both cells. Junctional permeability to ATP will be measured by the change in photon emission of an intracellular luciferin-luciferase system by injecting ATP into one cell of a partially HEP-depleted cell pair. Free radicals will be injected intracellularly into otherwise unperturbed cell pairs and the effects on gj assessed.